The present invention relates to a microbiological method for the manufacture of the antibiotic pseudomonic acid A (mupirocin).
Pseudomonic acid A, also known as mupirocin, is an antibiotic that has a growth inhibiting effect mainly against Gram positive bacteria (e.g. Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae) and some Gram negative bacteria (e.g. Haemophilus influenzae, Neisseria gonorrhoeae) [A. Ward, D. M.Campoli-Richards, Drugs 32, 425-444 (1986)] and its minimal inhibiting concentration is in the range of 0.02-0.5 mg/dm3. Pseudomonic acid A, by inhibiting the isoleucine-tRNA synthase enzyme, affects the peptide synthesis of pathogen bacteria [J. Hughes and G. Mellows, Biochem. J. 191, 209-219 (1980)]. An advantageous feature of this antibiotic is that it has very low toxicity both for humans and animals and it is negative in the Ames test. Pseudomonic acid A is presently used in human therapy, in various formulations, for the treatment of skin infections (e.g. impetigo, pyoderma), nose and external ear infections, acne, burns, eczema, psoriasis, in case of ulceration for treatment of secondary infections, and for prevention of hospital infections.
The chemical structure of pseudomonic acid A was determined to be 9-{4[5S(2S,3S-epoxy-5S-hydroxy-4S-methylhexyl)-3R,4R-dihydroxy-tetrahydropyran-2S-yl]-3-methylbut-2(E)-enoyloxy}nonanoic acid [E. B. Chain and G. Mellows, J. C. S. Chem. Comm. 847-848 (1974); R. G. Alexander, J. P. Clayton, K. Luk, N. H. Rogers, T. J. King, J. C. S. Perkin I 561-565 (1978)], as depicted by formula (I): 
It is known that Pseudomonas fluorescens is able to produce the pseudomonic acid A. According to the British Patent No. 1,395,907, the Pseudomonas fluorescens NCIB 10586 strain is able to biosynthesize the pseudomonic acid complex consisting of pseudomonic acid A and its isomer being a double bond in the cis position between the carbon atoms C2 and C3 and pseudomonic acid B. The ratio of the components is 4.5:4.5:1. According to the Japanese patent application No. 52-70083, however, the Pseudomonas fluorescens Y-11633 strain is able to biosynthesize the pseudomonic acid complex consisting of the pseudomonic acid A, pseudomonic acid B and further two components with unknown structures in the ratio of 9:0.5:0.5.
The present invention is directed to a procedure for the preparation of pseudomonic acid A comprising cultivating on a medium comprising at least one organic nitrogen or carbon source, in submerged aerated conditions, a Pseudomonas bacterium strain capable of the biosynthesis of pseudomonic acid A, and fermentation of the Pseudomonas culture such that pseudomonic acid A is formed. Preferably the Pseudomonas bacterium strain is Pseudomonas sp. bacterium strain No. 19/26 deposited under accession No. NCAIM(P)B 001235 in the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary, or its pseudomonic acid A-producing mutant or variant.
The present invention also is directed to a Pseudomonas culture capable of biosynthesizing pseudomonic acid A in submerged aerated conditions, consisting essentially of a novel Pseudomonas sp. bacterium strain No. 19/26.
The present invention further is directed to a biologically pure culture of a novel Pseudomonas sp. bacterium strain No. 19/26.